Pathologists have debated if fat or inflammation are necessary precursors of fibrosis for over a century. Importantly, we have very recently adapted the enteral alcohol delivery system developed for the rat by Tsukamoto and French to short-term studies in the mouse so that gene knockouts can be studied. Using the TNFR1 knockout in our new model, we obtained unequivocal evidence for the involvement of TNFalpha in early alcohol-induced liver injury. Mechanisms of alcohol-induced fibrosis remain less clear, and the purpose of this proposal is to use this new enteral model developed by us with knockout mice and gene delivery technqiues in long-term studies of alcohol- induced liver injury to fill important gaps in our knowledge. The unifying hypothesis we will test here using knockout technology is that inflammation but not fat is required for alcohol-induced fibrosis. We propose that this action is via oxidants. Initially, in Aim 1 we will optimize, characterize and validate a long-term mouse model of enteral alcohol delivery by determining pathology including fibrosis in wild-type mice which are background strains for knockouts selected for study. In Aim 2, we will take advantage of our pilot observation that the TNF receptor 1 knockout developed neither fat nor inflammation to test the hypothesis that fibrosis requires both. Next, we will study the effect of long-term enteral alcohol on fibrosis in knockouts which will exhibit fat but not inflammation (ICAM-1 knockouts) and inflammation but not fat (protein kinase A RIIbeta subunit knockouts). Finally, in Aim 3, we will use gene delivery of TGFbeta to TNF-R1 knockouts (i.e., no fat or inflammation) to test the hypothesis that alcohol-induced fibrosis is due to the pivotal cytokine TGFbeta alone. Next, antisense and dominant negatives to TGFbeta will be used to block fibrosis in wild-type mice. This work is timely and exciting since it will utilize our new enteral feeding model in the mouse and will allow us and others to investigate the roles of specific proteins and enzymes in alcohol-induced liver disease using the power of gene knockouts and gene delivery technology. This will position us uniquely to provide unequivocal new information and fill critical gaps in our knowledge on mechanisms of long-term alcohol-induced liver injury and set the stage for clinical trials.